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sc-12-0417_sm_SupplFigure1.tif84KSuppl. Figure 1. Experimental Design – atherosclerosis regression in the Reversa mouse model. Female and male Reversa mice were weaned at 21 days of age and separated into three groups. All three groups were fed a Western diet for 84 days. The first group, atherosclerosis-resistant (negative control), was given 4 intraperitoneal injections of pI-pC immediately after weaning. In the second group, the atherosclerosisprone group (−pI-pC), hepatic lipoprotein production remained unaltered as these mice were not injected with pI-pC. In the third group Mttp was inactivated after 84 days on the Western diet. We named this latter group of mice the atheroregressing group (+pI-pC). Atherosclerosis burden was monitored in all three groups immediately after the last (4th) pI-pC injection (day 0) or 16, 42 or 70 days after Cre-dependent Mttp inactivation in the atheroregressing group. Yellow block area, mice fed the Western diet; Atheroregression, in the +pI-pC group only; d, days.
sc-12-0417_sm_SupplFigure2.tif216KSuppl. Figure 2. Foamy macrophages are the main cell type populating atherosclerotic plaques in Reversa mice. Mice were fed the Western diet for 84 days and sacrificed. Aortic cross-sections were stained with Oil Red O and counterstained with hematoxylin (left). Sections were also stained with Moma 2-directed monoclonal Ab to identify lesional foamy macrophages (green, right).
sc-12-0417_sm_SupplFigure3.tif389KSuppl. Figure 3. Normalization of plasma lipids triggers regression of plaques. Reversa mice fed the Western diet were sacrificed immediately after the last pI-pC injection (day 0, base level) or 16, 42 and 70 days after Mttp inactivation. Aortas were removed, fixed and sectioned. Representative Oil Red O staining demonstrating the entire aortic root cross-sections of aortas from atherosclerosis-prone (−pI-pC) or atheroregressing (+pI-pC) mice.
sc-12-0417_sm_SupplFigure4A.tif71KSuppl. Figure 4. AMD3100 triggers leukocytosis and neutrophilia but does not alter plasma cholesterol. Hypercholesterolemia was reversed after 84 days on the Western diet and atheroregressing mice were treated with AMD3100 or saline for a total of 70 days. Peripheral blood was collected and total leukocyte and neutrophil numbers and plasma cholesterol levels were examined. A, Leukocyte and neutrophil numbers were determined with Hemavet. B, Fasting total plasma cholesterol and fasting plasma levels of LDL, HDL and triglycerides were measured using the colorimetric Cholesterol quantification kit.
sc-12-0417_sm_SupplFigure4B.tif78KSuppl. Figure 4. AMD3100 triggers leukocytosis and neutrophilia but does not alter plasma cholesterol. Hypercholesterolemia was reversed after 84 days on the Western diet and atheroregressing mice were treated with AMD3100 or saline for a total of 70 days. Peripheral blood was collected and total leukocyte and neutrophil numbers and plasma cholesterol levels were examined. A, Leukocyte and neutrophil numbers were determined with Hemavet. B, Fasting total plasma cholesterol and fasting plasma levels of LDL, HDL and triglycerides were measured using the colorimetric Cholesterol quantification kit.
sc-12-0417_sm_SupplFigure5A.tif134KSuppl. Figure 5. Evaluation of circulating EPCs and characterization of bone marrow Tie2-GFP+ EPCs. A, Circulating mononuclear cells obtained from atherosclerosis-prone (−pI-pC) or atheroregressing mouse treated with saline (+pI-pC) or AMD3100 (+pI-pC + AMD3100) were stained and analyzed as detailed in Materials and Methods. Fluorescence staining profiles of Flk1+ cells (left) and expression of CD34 and CD133 in the Flk1+ populations (right) are shown. Numbers in the upper right corner indicate percent of cells with the indicated phenotype. B, Bone marrow was collected from long bones of Tie2-GFP+ donor mice and EPCs isolated by negative selection and sorting were cultured ex vivo for 4 days. A, FACS plot shows representative Lin-GFP+ cells (GFP) co-stained with CD133, Sca-1-, Flk1-specific monoclonal antibodies, or with isotype rat IgG2b antibody. C, Representative images of Lin-GFP+ bone marrow cells (GFP, green) that were prior to adoptive transfer labeled with TRITC-Lectin (red) or evaluated for the uptake of Dil-AcLDL (red), and stained with DAPI (blue). Original magnification ×200.
sc-12-0417_sm_SupplFigure5B.tif153KSuppl. Figure 5. Evaluation of circulating EPCs and characterization of bone marrow Tie2-GFP+ EPCs. A, Circulating mononuclear cells obtained from atherosclerosis-prone (−pI-pC) or atheroregressing mouse treated with saline (+pI-pC) or AMD3100 (+pI-pC + AMD3100) were stained and analyzed as detailed in Materials and Methods. Fluorescence staining profiles of Flk1+ cells (left) and expression of CD34 and CD133 in the Flk1+ populations (right) are shown. Numbers in the upper right corner indicate percent of cells with the indicated phenotype. B, Bone marrow was collected from long bones of Tie2-GFP+ donor mice and EPCs isolated by negative selection and sorting were cultured ex vivo for 4 days. A, FACS plot shows representative Lin-GFP+ cells (GFP) co-stained with CD133, Sca-1-, Flk1-specific monoclonal antibodies, or with isotype rat IgG2b antibody. C, Representative images of Lin-GFP+ bone marrow cells (GFP, green) that were prior to adoptive transfer labeled with TRITC-Lectin (red) or evaluated for the uptake of Dil-AcLDL (red), and stained with DAPI (blue). Original magnification ×200.
sc-12-0417_sm_SupplFigure5C.tif368KSuppl. Figure 5. Evaluation of circulating EPCs and characterization of bone marrow Tie2-GFP+ EPCs. A, Circulating mononuclear cells obtained from atherosclerosis-prone (−pI-pC) or atheroregressing mouse treated with saline (+pI-pC) or AMD3100 (+pI-pC + AMD3100) were stained and analyzed as detailed in Materials and Methods. Fluorescence staining profiles of Flk1+ cells (left) and expression of CD34 and CD133 in the Flk1+ populations (right) are shown. Numbers in the upper right corner indicate percent of cells with the indicated phenotype. B, Bone marrow was collected from long bones of Tie2-GFP+ donor mice and EPCs isolated by negative selection and sorting were cultured ex vivo for 4 days. A, FACS plot shows representative Lin-GFP+ cells (GFP) co-stained with CD133, Sca-1-, Flk1-specific monoclonal antibodies, or with isotype rat IgG2b antibody. C, Representative images of Lin-GFP+ bone marrow cells (GFP, green) that were prior to adoptive transfer labeled with TRITC-Lectin (red) or evaluated for the uptake of Dil-AcLDL (red), and stained with DAPI (blue). Original magnification ×200.
sc-12-0417_sm_SupplFigure6.tif203KSuppl. Figure 6. Bone marrow EPCs augment plaque resolution. Reversa mice fed the Western diet for 84 days in which hypercholesterolemia was reversed were either treated with AMD3100 or saline, or received adoptive transfer of Tie2-GFP+ bone marrow EPCs (GFP+EPCs) three times, 10, 20 and 30 days after Mttp inactivation. Mice in all three groups were sacrificed 70 days after the last pI-pC injection and atherosclerosis burden was assessed. Representative Oil Red O staining showing the entire aortic root cross-sections.
sc-12-0417_sm_SupplFigure7.tif373KSuppl. Figure 7. EPC treatment upregulates the chemokine CXCL1 in regressing plaques. Atherosclerosis-prone (−pI-pC) or atheroregressing (+pI-pC) mice treated with saline or with Tie2-GFP+ bone marrow EPCs were sacrificed 70 days after Mttp inactivation. Aortas were removed, aortic roots were excised and frozen, and cryosections were stained with CXCL1, ICAM-1 or VCAM Ab and mounted in DAPIcontaining medium. Representative images of the aortic cross-sections from each group are shown.
sc-12-0417_sm_SupplFigure8.tif742KSuppl. Figure 8. Tie2-GFP+ EPCs in addition to vascular endothelium also populate spleen. Atheroregressing Reversa mice treated with either saline or AMD3100, or with Tie2-GFP+ bone marrow EPCs were sacrificed 70 days after Mttp inactivation and lungs, liver and spleen were collected, frozen, and cryosections were stained with primary rat anti-mouse CD31 or control rat IgG2a antibody and secondary goat anti-rat IgG Alexa Fluor 568 antibody to identify differentiated endothelial cells and mounted in DAPI-containing medium. The only organ that Tie2-GFP+ bone marrow EPCs were detected in addition to the vasculature was spleen. Representative images of the spleen section from each group. DAPI (blue), GFP (green), CD31 (red), red + green emission overlap = yellow.

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